1. Field of the Invention
The present invention relates generally to the fields of molecular biology and nucleic acid chemistry. More specifically, it relates to methods for the polymerase chain reaction amplification of long nucleic acid sequences.
2. Description of Related Art
The polymerase chain reaction (PCR), a powerful tool for the amplification of nucleic acid sequences, is disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188, each incorporated herein by reference. In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to complementary strands and flank the region of interest in the target DNA. A repetitive series of reaction steps involving template denaturation, primer annealing, and the extension of the annealed primers by a DNA polymerase results in the geometric accumulation of a specific fragment whose termini are defined by the 5' ends of the primers. PCR is capable of producing a selective enrichment of a specific DNA sequence by a factor of 10.sup.9. The PCR method is also described in Saiki et al., 1985, Science 230:1350, incorporated herein by reference.
PCR has been widely applied in molecular biology, molecular evolution, medical genetics, population genetics, forensic biology, and genome mapping and sequencing projects. However, current PCR are limited in the size of the region of DNA that can be amplified reliably.
Attempts to overcome the length limitations of PCR are reported in Glukhov et al., 1991, Molek. Biol. 25:1602-1610; Kainz et al., 1992, Anal. Biochem. 202:46-49; Ohler and Rose, 1992, PCR Meth. Applic. 2:51-59; Ponce and Micol, 1992, Nucl. Acids Res. 20:623; and Rychlik et al., 1990, Nucl. Acids Res. 18:6409-6412; each incorporated herein by reference. Although amplifications of 5-15 kb sequences were achieved, the reported yields of the longer products were low.
PCR methods capable of amplifying long nucleic acid sequences would facilitate genomic mapping and sequencing as well as molecular cloning through the amplification of long, low-copy insert material, and by making possible the assembly of larger recombinant constructions in PCR-based mutagenesis. There remains a need for methods that will enable PCR amplification of targets of at least 25 kb with high yields.